Aragonite

Studies were conducted at three shallow submarine volcanic CO₂ seeps and three adjacent control sites with similar geomorphology, seawater temperature and salinity, in Milne Bay Province, PNG [7 ]. The three seep locations (Dobu, Esa'Ala and Upa Upasina) are located along an active tectonic fault line, and almost pure CO₂ gas has been streaming through the reef substrata for an unknown period of time (confirmed for approx. 70 years, but possibly much longer [7 ]), resulting in locally reduced seawater pH. Areas of intense seeping with a median pH_total < 7.7 (approx. 1100 µatm pCO₂) were not included in the surveys, because no reef development is found beyond this apparent threshold. Mean hard coral cover was similar at the seeps compared with the adjacent control sites (33% ± 2.4 s.e. versus 31% ± 3.4 s.e.). However, at high CO₂, the cover of massive Porites corals is twice that of the control sites, the cover of structurally complex corals with branching, foliose and tabulate growth forms is reduced threefold, and coral diversity is reduced by 39% [7 ]. Seawater temperature and salinity are similar between seep and control sites [7 ].
A total of 968 discrete seawater samples of pH were taken at the six sites from approximately 0.5 m above the benthos (see electronic supplementary material). The data include all samples collected between 2010 and 2012, representing a range of tidal, irradiance and wave conditions to characterize the ranges encountered by the organisms. The samples were immediately analysed for pH, temperature and salinity, and a subset of 450 samples were preserved for later determination of total alkalinity and dissolved inorganic carbon. Other relevant seawater carbonate parameters (aragonite saturation state, dissolved inorganic carbon, pCO₂) were calculated from pH, total alkalinity, dissolved inorganic carbon, salinity and temperature using the R program Seacarb v. 2.4.8 [25 ].

To estimate site specific net CaCO₃ accretion rates and to relate calcification/dissolution to natural variability in pH, the SeaFET sensors were co-located with Calcification/Accretion Units (CAUs) (Fig. S1). A CAU consisted of a pair of roughly sanded PVC plates (10×10 cm) stacked 1 cm apart and plate pairs (N = 5 per site) were affixed to reef pavement at each site (>0.5 m apart and 10 cm above the substrate) using stainless steel rods and marine epoxy. Immediately after collection, all four surfaces of each CAU were photographed to determine early-successional community structure using the image analysis software PhotoGrid 1.0 (25 stratified random points analyzed per surface); organisms were sorted into ecological functional groups to look for patterns structuring the communities on the benthos and on the CAUs. Plates were then preserved in 8% formalin for subsequent measures of calcification rates.
To quantify the mass of CaCO₃ accumulated on a CAU, the plates were dried to a constant weight at 60°C and then weighed. Subsequently, CAUs were submerged in 5% HCl for 48 hrs or until all CaCO₃ had dissolved. The remaining fleshy tissue was scraped onto pre-weighed 11 µm cellulose filter paper, vacuum filtered, dried, and weighed to determine the difference in calcified to fleshy biomass on CAU surfaces. Finally, the acidified, scraped, and dried CAU plates were weighed. Calcimass was determined by subtracting the weight of the fleshy tissue and PVC plates from the total mass of the CAU. For all taxa recruiting to CAUs, the polymorph of CaCO₃ deposited is known. Thus, the relative net accretion for each polymorph (calcite, aragonite, high Mg calcite) was calculated by multiplying the net calcification rate by the relative abundance of each calcifying taxa of known mineralogy.

Prior studies indicate that borate rather than boric acid is the predominant species occupying the lattice position normally taken up by the carbonate ion52 in calcifiers that precipitate aragonite skeletons. Although there are a number of reaction pathways through which this substitution could occur19 20 (link), it is likely to involve de-protonation of the borate species to create a divalent base ion with the same charge as that of the carbonate ion species (−2), to preserve the charge neutrality of the growing crystal:

The partitioning of borate versus carbonate into aragonite is thus likely to be sensitive to solution pH10 19 20 (link). Here the relevant partition coefficient K_D relating the molar ratio of to the concentrations of the carbonate and borate species in the precipitating solution is determined using:

Holcomb et al.19 conducted experiments quantifying the ratio of boron to calcium in aragonite precipitated inorganically under a wide range of carbonate chemistries (including pH) and total DIC and boron concentrations, as well as conditions of pH and DIC appropriate to those in the calcifying fluid of corals. Furthermore, Holcomb et al.19 also showed the close relationships between B/Ca, CO₃²⁻ and K_D based on substitution reactions between B(OH)₄⁻ and CO₃²⁻. Re-analysing the Holcomb et al.19 data, we find (Fig. 5) that the observed K_D as defined in equation (4) shows the expected decrease as a function of the concentration of total active protons within the precipitating solution.
Thus, using the definition of K_D from equation (4) and its dependency on pH_cf as given by the inorganic data of Holcomb et al.19 , we can now calculate the concentration of carbonate ions within the calcifying fluid (that is, [CO₃²⁻]_cf from measurements of (B/Ca)_carb and pH_cf, the latter derived from the skeletal boron isotopic ratio (δ¹¹B_carb). We further assume that [B_T]_cf is equal to the total concentration of boron of ambient seawater and only a function of seawater salinity ([B_T]_cf=[B_T]_sw at salinity=35). We therefore have:

Where K_D=0.00297exp(−0.0202 [H⁺]_T and for typical calcifying fluid pH_cf values K_D ∼0.0027, an order of magnitude higher than a previous estimate20 (link). The concentration of DIC within the calcifying fluid is then calculated from the measured pH_cf (equation 1) and (equation 2) values using the programme CO₂SYS provided by Lewis and Wallace53 , with the carbonate species dissociation constants of Mehrbach et al.54 as re-fitted by Dickson and Millero55 , the borate and sulfate dissociation constants of Dickson51 56 , and the aragonite solubility constants of Mucci57 . We also note that our use of a reliable experimentally determined K_D is now consistent with substitution of borate with carbonate ion, rather than the previously inferred20 (link) substitution with bicarbonate ion, the latter assumption effectively negating the role of carbonate saturation state on calcification.

Cassiopea sp. were collected by hand while wading and snorkeling at our field locations in Lido Key, Florida, and Grassy Key, Florida. Due to the uncertain phylogeny of Cassiopea and the visual similarity between species, animals were not identified beyond genus level, although the two species recorded from Florida are C. andromeda and C. frondosa^{11 (link)}. These jellyfish were held at the University of South Florida in Tampa, Florida, in a 300 L closed-loop recirculating aquarium system. The salinity in this tank was maintained between 33 and 39‰ with Instant Ocean aquarium salt, and included a substrate of aragonite sand and high-intensity metal halide lighting on a 12:12 light cycle. Water temperature was maintained at ca. 28 °C over the duration of experiments.

The surface sample (of thick slides) was polished with alumina of 1 µm, 0.3 µm, and 0.05 µm and finally polished with colloidal silica (0.05 µm). Before analysis, samples were coated with a thin layer (ca. 2 nm) of carbon using a high vacuum coater. The EBSD study was carried out with Oxford NordlysMax detector mounted on a scanning electron microscope JEOL JSM-6610LV at the Institute of Materials Engineering, Łódź University of Technology. EBSD data were collected with AztecHKL software at high vacuum, 20 kV, large probe current, and 20 mm of working distance. EBSD patterns were collected at a resolution of 0.22 μm step size for crystallographic maps using the unit cell settings characteristic of aragonite and calcite as follows^{58 (link),59 (link)}: “Pmcn” symmetry and a = 4.96 Å, b = 7.97 Å, and c = 5.75 Å estimated for Favia coral using X-ray powder diffraction with synchrotron radiation (43) and a = b = 4.99 Å, and c = 17.06 Å, respectively. The EBSD data are represented in this study by crystallographic maps, phase images, and the pole figures, which represent the stereographic projection of crystallographic planes in reference to the (100), (010), (001) and (222) aragonite planes. Orientation images and the pole figures were created using MTEX open source plugin for Matlab program (https://mtex-toolbox.github.io/). To eliminate combination of red and green colors and create images more accessible for color-blind users we selected BungeColorKey palette from MTEX (the outcome was tested using Coblis, the Color Blindness Simulator at https://www.color-blindness.com/coblis-color-blindness-simulator/).

Two types of experimental methods were set up: (1) solution experiments and (2) replacement experiments. The combination of these two experimental settings and the studied temperature ranges allowed us to gain an in-depth picture on the kinetics and the governing factors during the crystallisation of Ce-bearing solids in the Ce–CO₃–H₂O system and provide detailed description for future material fabrication.
In the solution experiments, 10 ml of 50 mM Ce-bearing aqueous (Milli-Q) solutions (pH ≈ 5.1) were added to 10 ml of 50 mM Na₂CO₃ solutions in 20 ml Teflon-lined stainless-steel autoclaves at different temperatures (35, 50, 80 °C) and at saturated water vapor pressures (Table 1). To get more insight into the interaction of Ce and carbonate ions and the effects of temperature and concentration, additional 10 ml Ce-bearing aqueous (Milli-Q) solution (pH ≈ 5.1) mixed with 10 ml of Na₂CO₃ solution experiments were pre-heated and placed in hydrothermal reactors at 80 °C with different molar ratios of 1 : 1, 3 : 4 and 1 : 2 (Table 1). The solid samples were taken carefully at increasing time intervals. The reaction products were chilled to room temperature and filtered through 0.2 μm polycarbonate membranes by using a vacuum filtration unit. The solids were then placed into an oven at 50 °C for 30 min to remove any excess water.
In the replacement setting, 0.1 g of calcite, dolomite, or aragonite with sizes of 0.5–1.0 mm were added to 50 ml of 50 mM Ce-bearing aqueous (Milli-Q) solutions (Ce(NO₃)₃·6H₂O; pH ≈ 5.1). The solutions each were prepared using cerium(iii) nitrate hexahydrate (Ce(NO₃)₃·6H₂O) reagents (Sigma-Aldrich; 99.99% trace metals basis). The solutions and solids were placed in 50 ml Teflon-lined stainless-steel autoclaves at different temperatures (50, 90, 165, and 205 °C) and saturated water vapor pressures (Tables 2 and 3). Solid samples were taken carefully at increasing time intervals which were then placed into an oven at 50 °C for 30 min to remove any excess water. Also, control experiments consisting only of Ce-bearing aqueous solutions in closed reactors were carried out at the same temperatures of the solution and replacement experiments.